JP2008500373A - Cerberus / Coco derivatives and their use - Google Patents

Abstract

The present invention relates to Cerberus / Dan / gremlin polypeptides or variants thereof for use in the treatment of diseases associated with myostatin, Nodal, and GDF-11. Preferred polypeptides are Coco or Kerberos derivatives.

Description

Related Applications This application claims the benefit of US Provisional Patent Application No. 60 / 575,062 filed May 27, 2004. All of the teachings of the above applications are incorporated herein by reference.

Background of the Invention Transforming growth factor-β superfamily proteins represent a large family of cytokines including TGF-β, activin, inhibin, bone morphogenetic protein (BMP), and Muellerian tube inhibitor (MIS) (for review, Massague et al., Trends Cell Biol. 7: 187-192, 1997 (see Non-Patent Document 1). These proteins contain a conserved C-terminal cystine knot motif and function as ligands for a diverse family of plasma membrane receptors. Members of the TGF-β family have a wide range of biological effects on a wide variety of cell types. For example, they control cell proliferation, differentiation, substrate production, and apoptosis. Many of them have important functions in pattern formation and tissue specification during embryonic development; in adults, they are involved in processes such as tissue repair and regulation of the immune system.

  The activity of TGF-β superfamily proteins is controlled by various means. One negative regulation of the BMP subfamily of proteins is through a relatively large family of so-called bone morphogenetic protein (BMP) antagonists / suppressors. These BMP inhibitors represent a subgroup of proteins that bind to BMP and prevent it from binding to their membrane receptors, thereby blocking BMP activity during development and morphogenesis.

  BMP inhibitors are based on structural analysis, especially the number of structurally conserved Cys residues in the characteristic “Cys knot” structure at the C-terminus: three groups of proteins: 8-membered, 9-membered, or It can be further classified as a 10-membered Cys knot BMP inhibitor. Eight-membered ring (CAN subfamily) repressors can be further classified into four subgroups based on the conserved configuration of additional cysteine residues-gremlin and PRDC, cerberus and coco, and dans (DAN), and USAG-1 and sclerostin. Orthologs of these human BMP antagonists in the genomes of several model organisms have also been identified and their phylogenetic relationships have been analyzed (Avsian-Kretchmer and Hsueh, Mol Endocrinol. 18 (1): 1- 12, 2004. Epub 2003 Oct 02 (Non-Patent Document 2), incorporated herein by reference).

  Myostatin or growth / differentiation factor 8 (GDF-8) also belongs to the transforming growth factor-β (TGF-β) superfamily (McPherron et al., Nature 387: 83-90 (1997)). )). The human myostatin gene has been cloned (Nestor et al. Proc. Natl Acad. Sci. 95: 14938-43 (1998)), and myostatin immunoreactivity is human in type 1 and type 2 fibers It has been reported that it can be detected in skeletal muscle. In terms of function, myostatin may play a role in negatively controlling skeletal muscle growth and development (Nestor et al., Supra).

  Studies with myostatin knockout mice provided the first evidence that myostatin is an important negative regulator of muscle development (McPherron et al., Nature 387: 83-90 (1997)). )). In myostatin null mice, the animals were significantly larger than wild type mice and had a large and extensive increase in skeletal muscle mass. In addition, two-lineage cattle characterized by increased muscle mass have mutations in the myostatin coding sequence (McPherron et al., Proc. Natl. Acad. Sci. 94: 12457-61 (1997)). Five)). Naturally occurring myostatin hypofunction mutations in human children are associated with total muscle hypertrophy and a family history of extraordinary power (Williams MS, N Engl J Med. 2004 Sep 2; 351 (10): 1030-1; author reply 1030-1 (Non-Patent Document 6)).

  In addition, it should be noted that the serum and intramuscular concentrations of immunoreactive myostatin are increased in HIV-infected patients with muscle atrophy compared to healthy individuals and have an inverse correlation with the lean mass index. These data support the hypothesis that myostatin is a negative regulator of skeletal muscle growth in adults and contributes to muscle atrophy in HIV-infected patients (Nestor et al., Supra).

  In view of the above findings, myostatin activity, particularly in individuals with muscular atrophy as a result of pathologies or disease states such as aging, autoimmune deficiency syndrome (AIDS), multiple sclerosis, and cancer, for example There is a need for a way to control. The present invention provides methods and compositions that can be utilized to help individuals with such muscular atrophy status and provides further insight into the regulation of myostatin gene expression.

US Provisional Patent Application No. 60 / 575,062 Massague et al., Trends Cell Biol. 7: 187-192, 1997 Avsian-Kretchmer and Hsueh, Mol Endocrinol. 18 (1): 1-12, 2004. Epub 2003 Oct 02 McPherron et al., Nature 387: 83-90 (1997) Nestor et al. Proc. Natl Acad. Sci. 95: 14938-43 (1998) McPherron et al., Proc. Natl. Acad. Sci. 94: 12457-61 (1997) Williams MS, N Engl J Med. 2004 Sep 2; 351 (10): 1030-1; author reply 1030-1

Summary of the InventionOne aspect of the present invention is the Kerberos, Coco, or Kerberos / Dan / Gremlin superfamily (in summary herein) for inhibiting Nodal, myostatin, and BMP function / signaling. Pharmaceutical preparations of other polypeptides derived from "CDG protein"). Exemplary preparations of the subject CDG polypeptides include variant Kerberos or coco sequences that substantially retain the binding affinity of the parent protein for nodal, myostatin, and / or another BMP (such as BMP-4). obtain. For example, the present invention provides a pharmaceutical preparation for inhibiting myostatin comprising a myostatin antagonist protein comprising (at least) a myostatin binding domain of a Kerberos / Dan / Gremlin polypeptide or variant thereof. The myostatin antagonist protein binds to and neutralizes one or more of nodal and / or myostatin. Preferably, the pharmaceutical preparation is substantially free of pyrogens so that it is suitable for administration as a human or veterinary therapeutic.

  These pharmaceutical preparations can be used, at least in part, to reduce the severity of a pathological condition characterized by abnormal amount, development, or metabolic activity of muscle or adipose tissue in a subject . For example, the pharmaceutical preparations of the present invention may comprise cachexia, anorexia, DMD syndrome, BMD syndrome, AIDS wasting syndrome, muscular dystrophy, neuromuscular disease, motor neuron disease, neuromuscular junction disease, and inflammatory myopathy Can be administered in an amount effective to prevent, ameliorate, or reduce the severity of debilitating disease. These pharmaceutical preparations are also at least in part a pathological condition characterized by an abnormal amount, development, or metabolic activity of ectopic ossification in tissues such as muscles, tendons, and ligaments; Osteoarthritis (OA), including the development of synovial hyperplasia; ovarian cancer; progressive ossifying fibrosis (FOP); atherosclerosis, especially in the early stages of atherogenesis in the vulnerable area It can also be used to reduce the severity of inflammatory reactions; and skull fusion.

  Another aspect of the present invention is specific for nodal and / or myostatin function without substantially compromising BMP (such as BMP-4) signaling (eg, does not bind to BMP-4 or other BMPs). Pharmaceutical preparations of CDG protein derivatives for inhibiting are provided. Exemplary preparations of this aspect of the invention include polypeptides comprising a Kerberos or coco N-terminal truncated or other fragment containing a cysteine core. These so-called “N-terminally truncated CDG derivatives” are used, at least in part, to reduce the severity of a pathological condition characterized by an abnormal amount, development, or metabolic activity of muscle or adipose tissue in a subject can do. For example, the pharmaceutical preparations of the present invention may comprise cachexia, anorexia, DMD syndrome, BMD syndrome, AIDS wasting syndrome, muscular dystrophy, neuromuscular disease, motor neuron disease, neuromuscular junction disease, and inflammatory myopathy Can be administered in an amount effective to prevent, ameliorate, or reduce the severity of debilitating disease.

  In certain embodiments, the myostatin inhibitor is a polypeptide comprising a myostatin binding domain of a CDG protein. For example, a Kerberos protein variant can be derived from human, mouse, or other species of Kerberos, including at least about 50%, 60%, 70%, 80%, 90%, 95 with human or mouse Kerberos protein. Human or mouse Kerberos variant sequences that share%, or 99% or more sequence similarity or identity, and substantially retain the binding affinity of wild-type Kerberos for myostatin are included. Similarly, coco protein variants can also be derived from human, mouse, or other species of coco, including human or mouse coco protein and at least about 50%, 60%, 70%, 80%, 90% , 95%, or 99% or more of sequence similarity or identity, and human or mouse coco variant sequences that substantially retain binding affinity for wild-type coco myostatin.

  In certain related embodiments, the myostatin inhibitor is a polypeptide comprising a myostatin binding domain of a CDG protein, and the polypeptide does not substantially bind to BMP-4 (and other BMPs). For example, the myostatin binding domain can be derived from human, mouse, or other species of N-terminal truncated Kerberos, including residues 106-119 of SEQ ID NO: 2 or 8 of US 2002/0164682 A1 ( (See below) and ends at any residue after 241 residues of SEQ ID NO: 2 or 8 of US 2002/0164682 A1, preferably US 2002/0164682 A1 Included are human or mouse Kerberos derivatives having amino acid residues ending at any residue between 241 and 267 of SEQ ID NO: 2 or 8 (residue numbers include residues at both ends) ).

For example, residues 106-119 of human cerberus are as follows:
PPGTQSLIQPIDGM

The 241 to 267 residues of human cerberus are as follows:

Also included are Kerberos-derived variant sequences such as, for example, Kerberos N-terminal truncated myostatin binding domain that retains myostatin binding activity but has lost other BMP binding activity. Variant sequences alter the selectivity of the inhibitor (e.g., with respect to GDF-8, GDF-11, or Nodal binding) and alter other binding properties (K _d , and / or K _on or K _off rates) for myostatin. It may be desirable as a method of altering or improving biodistribution or half-life in vivo or during storage.

In certain preferred embodiments, the Cerberus polypeptide (full length or N-terminal truncated) containing myostatin binding domain, 1 [mu] M following K _d, more preferably 100 nM, 10 nM or even in the following K _d 1 nM, myostatin Combine with.

である。 In certain related embodiments, the myostatin inhibitor is a polypeptide comprising a myostatin binding domain of a coco protein such as the human coco protein shown in FIG. 3 or GenBank Accession No. 2227329. An exemplary human coco protein sequence is:

It is.

In certain preferred embodiments, here a polypeptide comprising a myostatin binding domain (full length or N-terminal truncated) is, 1 [mu] M following K _d, more preferably 100 nM, 10 nM or even in the following K _d 1 nM, myostatin Combine with.

In certain embodiments, the myostatin binding domain, in addition to the myostatin binding domain, is one of in vivo stability, in vivo half-life, attachment / administration, tissue localization or distribution, protein complex formation, and / or purification. Or part of a fusion protein comprising one or more polypeptide moieties that enhance the plurality. For example, the fusion protein can include an immunoglobulin Fc domain. The fusion protein can include a purified subsequence such as an epitope tag, a FLAG tag, a polyhistidine sequence, or a GST fusion.

  In certain embodiments, the myostatin binding domain is a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid bound to a lipid moiety, or an amino acid bound to an organic derivatizing agent. A part of a protein containing one or more modified amino acid residues.

In certain embodiments, the variant CDG polypeptide is selective for myostatin binding and inhibition, eg, compared to GDF-11 and / or Nodal. For example, the variant CDG polypeptide is at least 2-fold lower than the dissociation constant (K _d ) for binding to GDF-11 and / or nodal with respect to myostatin binding, even more preferably at least 5, 10, 100, or even _May have a K _d that is 1000 times lower. Whether due to binding kinetics or biodistribution, this variant CDG polypeptide also has a myostatin activity or specific physiological consequence (promoting muscle growth, promoting bone density, or adipocyte differentiation). for induction, etc.), inhibitors with GDF-11 and / or at least 2 fold less than the EC ₅₀ for in inhibiting nodal activity, even more preferably at least 5, 10, 100 or even 1000-fold lower EC _50, As such, it can be selected based on relative in vivo capabilities.

In certain embodiments, the variant CDG polypeptide is selective for myostatin binding and inhibition compared to other BMP proteins, such as, for example, BMP-4. For example, the variant CDG polypeptide is at least 2-fold lower, even more preferably at least 5, 10, 100, or even 1000-fold lower with respect to myostatin binding than the dissociation constant (K _d ) when binding to BMP-4. It may have _Kd . Whether due to binding kinetics or biodistribution, this variant CDG polypeptide also has a myostatin activity or specific physiological consequence (promoting muscle growth, promoting bone density, or adipocyte differentiation). for induction, etc.), at least 2-fold lower than the EC ₅₀ for in inhibiting BMP-4 activity, as inhibitors even more preferably at least 5, 10, 100 or even 1000-fold lower EC _50,, Selection can be based on relative in vivo capacity.

In certain preferred embodiments, the variant CDG polypeptide binding domains, 1 [mu] M or less less K _d, more preferably 100 nM, 10 nM or even, binds to myostatin at 1 nM or less of the K _d.

  In general, the myostatin inhibitor preparation is suitable for use in human patients. In a preferred embodiment, the preparation of variant CDG polypeptide is substantially free of pyrogens so that it is suitable for administration to human patients.

  In other embodiments, the subject variant CDG polypeptides can be administered to non-human animals, particularly other mammals. For example, the compounds of the invention can be administered to chickens, turkeys, livestock animals (sheep, pigs, horses, cows, etc.), pet animals (eg, cats and dogs), or promote growth and protein / fat Can help in aquaculture to improve the ratio. To further illustrate, this variant Kerberos polypeptide should be used to improve the quality of carcass, promote the growth of meat-producing animals or increase feed efficiency, or increase the milk production of dairy cows. Can do.

  Another aspect of the present invention relates to a packaged pharmaceutical comprising a pharmaceutical preparation of the variant CDG polypeptide described herein and a label or instructions for use in promoting muscle tissue growth in human patients. .

  Yet another aspect of the invention includes pharmaceutical preparations of the variant CDG polypeptides described herein, and labels or instructions for use by veterinarians to promote muscle tissue growth in non-human mammals. It relates to packaged pharmaceuticals.

  Another aspect of the invention pertains to methods of inhibiting myostatin signaling in vivo by administering one or more pharmaceutical preparations of the subject variant CDG polypeptides. The method can be used to promote muscle growth, promote adipogenic differentiation, and / or promote bone growth or bone mineralization in a human patient or non-human animal.

  In certain embodiments, the treatment methods of the invention are used to reduce, at least in part, the severity of a pathological condition characterized by abnormal amount, development, or metabolic activity of muscle or adipose tissue in a subject. be able to. For example, the pharmaceutical preparations of the present invention may comprise cachexia, anorexia, DMD syndrome, BMD syndrome, AIDS wasting syndrome, muscular dystrophy, neuromuscular disease, motor neuron disease, neuromuscular junction disease, and inflammatory myopathy Can be administered in an amount effective to prevent, ameliorate, or reduce the severity of debilitating disease.

  Exemplary muscular dystrophies that can be treated with therapies containing the present myostatin include the following: Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreis muscular dystrophy (EDMD), limb girdle Muscular dystrophy (LGMD), facial scapulohumeral muscular dystrophy (FSH or FSHD) (also known as Landsy Degerine), myotonic dystrophy (MMD) (also known as Steinert's disease), oropharyngeal muscular dystrophy (OPMD) ), Distal muscular dystrophy (DD), and congenital muscular dystrophy (CMD).

  Exemplary motor neuron diseases that can be treated using therapies containing this myostatin include: amyotrophic lateral sclerosis (ALS) (also known as Lou Gehrig's disease), infant progression Spinal muscular atrophy (SMA, SMA1, or WH) (type 1 SMA, also known as Weldnig Hoffmann), intermittent spinal muscular atrophy (SMA or SMA2) (also known as type 2 SMA), Juvenile spinal muscular atrophy (SMA, SMA3, or KW) (type 3 SMA, also known as Kugelberg Welander), medullary muscular atrophy (SBMA) (also known as Kennedy disease and X-linked SBMA) ), And adult spinal muscular atrophy (SMA).

  Exemplary inflammatory myopathy that can be treated with therapies containing the present myostatin include: dermatomyositis (PM / DM), polymyositis (PM / DM), and inclusion body myositis ( IBM).

  Exemplary neuromuscular junction diseases that can be treated with therapies containing the present myostatin include the following: myasthenia gravis (MG), Lambert Eaton syndrome (LES), and congenital myasthenia Syndrome (CMS).

  Exemplary muscle diseases resulting from endocrine abnormalities that can be treated with therapies containing this myostatin include the following: hyperthyroid muscle disease (HYPTM) and hypothyroid muscle disease (HYPOTM) ).

  Exemplary peripheral neurological diseases that can be treated using therapies that include the present myostatin include: Charcot-Marie-Tooth disease (CMT), DeJerrine-Sottus disease (DS), and Friedreich ataxia (FA).

  Other exemplary myopathy that can be treated using therapies that include the present myostatin include: congenital myoto (MC), congenital paramyotonia (PC), central core disease (CCD), Nemarine myopathy (NM), tubule myopathy (MTM or MM), and periodic paralysis (PP).

  Exemplary metabolic disorders of muscle that can be treated with therapies that include this myostatin include the following: phosphorylase deficiency (MPD or PYGM), acid maltase deficiency (AMD), phosphofructokinase deficiency Disease (PFKM), debranching enzyme deficiency (DBD), mitochondrial myopathy (MITO), carnitine deficiency (CD), carnitine palmitoyltransferase deficiency (CPT), phosphoglycerate kinase deficiency (PGK), phosphoglycerin Acid mutase deficiency (PGAM or PGAMM), lactate dehydrogenase deficiency (LDHA), and myoadenylate deaminase deficiency (MAD).

  The present invention can also be used to prevent, ameliorate, or reduce the severity of metabolic diseases, such as in the treatment of obesity or type II diabetes. To further illustrate, the present variant CDG polypeptide preparation can be used to reduce a subject's body fat ratio.

  In yet another aspect, the variant CDG polypeptide preparation treats or prevents congestive heart failure; reduces age-related weakness; increases bone density (such as for the treatment of osteoporosis) or fracture repair Treat growth retardation, treat physiological short stature, reduce protein catabolism such as after major surgery; reduce protein loss due to chronic disease; promote wound healing; burn patients or major surgery Can be used as part of methods such as maintaining recovery of skin thickness; maintaining metabolic homeostasis and renal homeostasis. Still other uses of this variant CDG polypeptide include the treatment of adults with growth hormone deficiency and the prevention of catabolic side effects of glucocorticoids.

  The pharmaceutical compositions can also be used as myostatin antagonists to treat many neurological disease states, including CNS injuries / disorders such as spinal cord injury and stroke, and PNS injuries / disorders.

  The present invention also contemplates the use of the present myostatin formulations in combination with one or more other compounds useful in an attempt to treat the diseases or indications described above. In these combinations, the therapeutic agent and the variant CDG polypeptide of the invention may be administered independently and sequentially, or may be administered simultaneously. Combination therapies for inhibiting bone resorption, preventing osteoporosis, reducing skeletal destruction, enhancing fracture healing, promoting bone formation, and increasing bone mineral density include bisphosphonates and It can be achieved by a combination of variant CDG polypeptides. Bisphosphonates having these utilities include, but are not limited to, alendronate, tiludronate, dimethyl-APD, risedronate, etidronate, YM-175, clodronate, pamidronate, and BM-210995 ( Ibandronate).

  The myostatin inhibitor can be used in combination with a mammalian estrogen agonist / antagonist. The term estrogen agonist / antagonist refers to a compound that binds to the estrogen receptor, inhibits bone turnover and prevents bone loss. In particular, an estrogen agonist is defined herein as a compound that can bind to an estrogen receptor site in mammalian tissue and mimic the action of estrogen in one or more tissues. An estrogen antagonist is defined herein as a compound that can bind to an estrogen receptor site in mammalian tissue and block the action of estrogen in one or more tissues. A variety of these compounds are described and referenced below, but other estrogen agonists / antagonists will be well known to those skilled in the art. Exemplary estrogen agonist / antagonists include droloxifene and related compounds (see US Pat.No. 5,047,431), tamoxifen and related compounds (see US Pat. No. 4,536,516), 4-hydroxy tamoxifen (US No. 4,623,660), raloxifene and related compounds (see 4 US Pat. No. 4,418,068), and idoxifene and related compounds (see US Pat. No. 4,839,155).

  The myostatin inhibitors can also be used in combination with one or more of the following agents: glutamate antagonists (including partial antagonists) such as riluzole and topiramate; polypeptide growth factors such as growth hormone ( GH) and insulin-like growth factor 1 (IGF-1), or drugs that increase the body's own production of neurotrophic factors, such as xaliproden; anti-inflammatory drugs, such as celecoxib (Celebrex) and other COX-2 inhibitors Antibiotics such as minocycline (minosin, dynacin) or other drugs that inhibit caspase enzymes; protein kinase C inhibitors such as tamoxifen (Norbadex); and various commercially available substances including vitamin E, coenzyme Q10, and creatine .

Detailed Description of the Invention
I. Overview Cerberus is expressed in the anterior mesoderm during development (Bouwmeester et al., Nature 382: 595-601, 1996; Piccolo et al., Nature 397: 707-10, 1999; Rodriguez et al., Nature 401: 243-51, 1999). The chicken ortholog caronte is involved in the left-right asymmetry of the chicken embryo (Rodriguez, supra). Cerberus functions as a multivalent growth factor antagonist in the extracellular space and inhibits signaling by BMP-4, Nodal, and Wnt (Belo et al., Genesis 26: 265-70, 2000). Mouse Kerberos binds to BMP protein and Nodal through independent sites (Piccolo, supra), but Xenopus kerberus also binds to Wnt protein and inhibits their action (Belo, supra). Cerberus has the unique property of inducing an ectopic head in the absence of trunk structure (Piccolo, supra). Cerebellar expression during gastrulation is activated by nodal-related signals in the endoderm and by Spemann organizer factors (Yamamoto et al., Dev Biol 257: 190-204, 2003).

  Cerberus orthologs are found in Xenopus tropicalis and Fugu rubripes but are not present in invertebrates. In Troughg, there is only one ortholog for Cerberus. All Cerberus orthologous genes have two exons; the first 8 amino acids of the cystine-knot domain are encoded by the 3 'end of the first exon, and the remainder of the motif is encoded by the second exon. In some orthologs, a putative protein cleavage site can be found upstream upstream of the cystine-knot domain.

  Coco is another member of the Kerberos / Dan family of proteins that inhibits Nodal signaling.

  In one, the present invention provides coco or cerberus derivatives for inhibiting nodal, GDF-11, and / or myostatin function. In certain embodiments, the coco and kerberus derivatives do not substantially impair BMP (such as BMP-4) signaling (e.g., bind to neither BMP-4 nor other BMPs), Nodal, GDF-11, And / or inhibit myostatin function. The cerberus derivatives can also be used to inhibit BMP (such as BMP-4) signaling.

  Exemplary preparations of the present invention include Kerberos polypeptide derivatives, including the N-terminal truncated form of Kerberos. These so-called “cerberus derivatives” may be used, at least in part, to reduce the severity of a pathological condition characterized by abnormal amount, development, or metabolic activity of muscle or adipose tissue in a subject. it can. For example, the pharmaceutical preparations of the present invention may comprise cachexia, eating disorders, DMD syndrome, BMD syndrome, AIDS wasting syndrome, muscular dystrophy, neuromuscular disease, motor neuron disease, neuromuscular junction disease, and inflammatory muscle. It can be administered in an amount effective to prevent, ameliorate, or reduce the severity of a debilitating disease such as a disease.

II. Definitions The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. In describing the compositions and methods of the present invention, and how to make and use them, certain terms are discussed below or elsewhere herein to provide additional guidance to the practitioner. The scope and meaning of any use of a term will be apparent from the specific context in which the term is used.

  “About” and “approximately” shall generally mean an acceptable degree of error in the quantity measured given the nature or accuracy of the measurement. Typically, exemplary degrees of error are within 20 percent (%) of a given value or range, preferably within 10%, more preferably within 5%.

  Or, particularly in biological systems, the terms “about” and “approximately” can mean a value within one order, preferably within five times, more preferably within two times a given value. The quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred even if not explicitly stated.

  The methods of the invention may include comparing sequences to each other, including wild type sequences and one or more variants / sequence variants. Such comparisons typically involve alignment of polymer sequences using, for example, sequence alignment programs and / or algorithms well known in the art (e.g., BLAST, FASTA, and MEGALIGN, to name a few). Including. In alignments where the mutation involves insertion or deletion of residues, the sequence alignment is represented by a “gap” (typically a dash or “A”) in the polymer sequence that does not include insertion or deletion residues. ) Can be easily understood by those skilled in the art.

  The term “homologous” is a “common origin of evolution” that includes, in all of its grammatical forms and spelling variants, proteins derived from the superfamily of the same species and homologous proteins from different species. Refers to the relationship between two proteins having Such proteins (and the nucleic acids that encode them) reflect their sequence similarity, whether in terms of percent identity or due to the presence of specific residues or motifs and conserved positions. Sequence homology.

  The term “sequence similarity” refers to the degree of identity or identity between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin in all of its grammatical forms.

  However, in general use and in this application, the term “homologous” can refer to sequence similarity when associated with an adverb such as “highly” and is associated with a common evolutionary origin It may or may not be.

  A nucleic acid molecule “hybridizes” to another nucleic acid molecule, such as cDNA, genomic DNA, or RNA, when a single-stranded form of the nucleic acid molecule can anneal to another nucleic acid molecule under appropriate conditions of temperature and solution ionic strength. (See Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The conditions of temperature and ionic strength determine the “stringency” of hybridization. For preliminary screening of homologous nucleic acids, low stringency hybridization conditions corresponding to a Tm (melting temperature) of 55 ° C., such as 5 × SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5 × SSC, 0.5% SDS can be used.

  Moderate stringency hybridization conditions correspond to higher Tm, for example 40% formamide with 5x or 6xSSC. High stringency hybridization conditions correspond to the highest Tm, for example 50% formamide, 5x or 6xSSC. SSC is 0.15M NaCl, 0.015M Na-citric acid.

  “High stringency conditions” include those hybridization conditions that allow hybridization of structurally related nucleic acids but do not allow hybridization of nucleic acids that are not structurally similar. Well understood in the field. The term “stringent” is a terminology understood by those of skill in the art to describe any of a number of selective hybridization and wash conditions that allow annealing of only highly complementary nucleic acids. .

Exemplary high stringency hybridization conditions correspond to a Tm that is about 20-27 ° C. lower than the melting temperature (Tm) of the DNA duplex formed in the about 1M salt. There are many equivalent procedures, and some general molecular cloning instructions describe the appropriate conditions for stringent hybridization and, in addition, hybrids that are expected to be stable under these conditions (Eg, Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6 or 13.6.6; or Sambrook, et al. ( ^{1989) Molecular Cloning, 2 nd ed} ., see 9.47-9.57 page of Cold Spring Harbor Press).

Hybridization requires that two nucleic acids contain complementary sequences, but depending on the stringency of hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The higher the degree of similarity or homology between two nucleotide sequences, the higher the _Tm value for hybrids of nucleic acids having those sequences. The relative stability of nucleic acid hybridization (corresponding to Tm height) decreases in the following order: RNA: RNA, DNA: RNA, DNA: DNA. For hybrids over 100 nucleotides in length, an equation has been derived for calculating the _Tm (see Sambrook et al., Supra, 9.51). For hybridization with shorter nucleic acids, ie oligonucleotides, the position of the mismatch becomes more important and the specificity is defined by the length of the oligonucleotide (see Sambrook et al., Supra, 11.8). The minimum length of a nucleic acid that can hybridize is at least about 10 nucleotides; preferably at least about 15 nucleotides; more preferably the length is at least about 20 nucleotides.

Unless otherwise stated, the term “standard hybridization conditions” refers to a T _{m of} about 55 ° C. and utilizes the conditions described above. In a preferred embodiment, T _m is 60 ° C .; in a more preferred embodiment, T _m is 65 ° C. In certain embodiments, “high stringency” is equivalent to the level of hybridization seen at 0.2 ° SSC at 68 ° C., 50% formamide, 4 × SSC at 42 ° C., or under either of these two conditions. Refers to hybridization and / or wash conditions under conditions that provide a level.

  Suitable hybridization conditions for oligonucleotides (eg, oligonucleotide probes or primers) are typically slightly different from conditions for full-length nucleic acids (eg, full-length cDNA) because of the low melting temperature of the oligonucleotide. Since the melting temperature of an oligonucleotide depends on the length of the associated oligonucleotide sequence, the appropriate hybridization temperature will vary depending on the oligonucleotide molecule used. Exemplary temperatures are 37 ° C (for 14 base oligonucleotides), 48 ° C (for 17 base oligonucleotides), 55 ° C (for 20 base oligonucleotides), and 60 ° C (23 base oligonucleotides). Against). Exemplary suitable hybridization conditions for oligonucleotides include washing in 6 × SSC, 0.05% sodium pyrophosphate, or other conditions that provide equivalent levels of hybridization.

  “Polypeptide”, “peptide”, and “protein” are used interchangeably to describe a chain of amino acids joined by chemical bonds referred to as “peptide bonds”. A protein or polypeptide, including an enzyme, may be “natural” or “wild type” meaning that it occurs in nature; or is it made, modified, or derived from a natural protein or another variant? Or “mutant”, “variant”, or “variant”, meaning that they are different or altered in some way.

  As used herein, the terms “cerberus / dan / gremlin superfamily protein” and “CDG protein” are both used to mean a protein family including cerberus, coco, and other related proteins.

ヒトケルベロスタンパク質(NCBI参照配列番号NP_005445)：

“Cerberus or Cerberus-like protein” also refers to mouse (NCBI reference SEQ ID NO: NP_034017) or human (NCBI reference SEQ ID NO: NP_005445) Kerberos protein (see also SEQ ID NO: 2 or 8 of US 2002/0164682 A1, respectively) These contents are incorporated herein by reference), and other proteins that share sequence homology with the highly conserved cysteine pattern of the C-terminal portion of mammalian Kerberos proteins, such as mammalian Kerberos and Kerberos-like Refers to protein. Exemplary amino acid sequences of Kerberos protein include the following: Mouse Kerberos protein (NCBI reference SEQ ID NO: NP_034017):

Human Cerberus protein (NCBI reference SEQ ID NO: NP_005445):

NM_009887.1(マウスケルベロスmRNA)。

Mouse and human cerberus are as disclosed in US 2002/0164682 A1 as SEQ ID NO: 1 and 7 (incorporated herein by reference).
NCBI Reference SEQ ID NO: NM — 005454.1 (Human Cerberus mRNA).

NM_009887.1 (mouse cerberus mRNA).

  Cerberus-related proteins also exist in other species, including family members in Xenopus and Drosophila, nematodes, zebrafish, and all mammals (e.g., rats, mice, and non-human primates) Is predicted. "Cerberus or Kerberos-like protein" includes variants of Kerberos protein that retain myostatin binding activity, eg, allelic variants or variants induced by mutagenesis or deletion, and fragments of Kerberos protein that retain myostatin binding activity Is also included. “Cerberus-like protein” is also used to indicate a family of proteins that share structural and / or functional similarities, including the proteins further described herein. Such proteins have significant sequence identity (e.g., at least about 50%, 60%, 70%) with human or mouse Kerberos proteins over the entire length of human or mouse Kerberos, or at least within the myostatin binding domain of human or mouse Kerberos. %, 80%, 90%, 95%, 99% or more). Cerberus-like proteins also include proteins having, under stringent conditions, a human or mouse Kerberos coding sequence, particularly an amino acid sequence encoded by a nucleic acid sequence that hybridizes with the coding sequence portion of the myostatin binding domain. The Kerberos derivative or Kerberos variant sequence may or may not lack an N-terminal BMP binding domain.

である。 “Coco or coco-like protein” refers to the mammalian coco protein of GenBank Accession 2273329 and other proteins that share sequence homology with the highly conserved cysteine pattern of the C-terminal portion of the mammalian coco protein. Refers to coco protein and related homologues. An exemplary amino acid sequence of human coco protein is:

It is.

The human cococode sequence is disclosed in GenBank accession 2273328 (incorporated herein by reference).

  Coco-related proteins are also present in other species, including Xenopus and Drosophila, nematodes, zebrafish, and family members in all mammals (e.g., rats, mice, and non-human primates). Is predicted. “Coco or coco-like protein” includes variants of natural coco proteins that retain myostatin binding activity, eg, allelic variants or variants induced by mutagenesis or deletion, and coco proteins that retain myostatin binding activity. Fragments are also included. “Coco-like protein” is also used to indicate a family of proteins that share structural and / or functional similarities, including the proteins further described herein. Such proteins have significant sequence identity with human coco proteins (e.g., at least about 50%, 60%, 70%, 80%, 90%, over the entire length of human coco, or at least within the myostatin binding domain of human coco. 95%, 99% or more) may be shared. Coco-like proteins also include proteins having an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions to the coding sequence of human coco, particularly the coding sequence portion of the myostatin binding domain. The coco derivative or coco variant sequence may or may not lack an N-terminal BMP binding domain.

  Except as otherwise noted, “cerberus (derivative) therapeutics” or grammatical variations thereof include full-length or N-terminal truncated kerberos therapeutics.

  As used herein, the term “cerberus or cerberus-like activity” refers to one or more of the activities exhibited by the mammalian cerberus-like proteins of the invention. In particular, “cerberus or cerberus-like activity” includes inducing and enhancing the formation, growth, proliferation, differentiation, and maintenance of neurons and / or related neurons and tissues such as brain cells, Schwann cells, glial cells, and astrocytes, And / or the ability to inhibit. “Cerberus or Cerberus-like activity” includes the ability to induce molecular markers of neuroendocrine or ectodermal tissue such as OTX2, H-CAM, MASH, chromogranin, and AP2, as well as neurons and / or brain cells, Schwann cells, glia Also included is the ability to induce the formation of cells and related neurons and tissues such as astrocytes. “Cerberus or cerberus-like activity” can also include the ability to control the interaction of ligands with their protein receptors. “Cerberus or cerberus-like activity” may further include the ability to control the formation, differentiation, proliferation and / or maintenance of other cells and / or tissues such as connective tissue, organs, and wound healing. In particular, “cerberus or cerberus-like activity” includes heart, spleen, liver, pancreas, stomach, kidney, lung, and brain cells and tissues, as well as osteoblasts and bones, chondrocytes and cartilage, tendons, epidermis and muscles. The ability to enhance and / or inhibit the formation, growth, proliferation, differentiation, and / or maintenance of can be included. “Cerberus or Kerberos-like activity” also includes the activity of Kerberos and Kerberos-like proteins in the assays described in the Examples and herein.

  The mouse and human Kerberos and Kerberos-like nucleotide sequences are as disclosed in US 2002/0164682 A1 as SEQ ID NO: 1 and 7 (incorporated herein by reference). See also NCBI reference SEQ ID NOs: NM_005454.1 (human) and NM_009887.1 (mouse).

である。 In certain related embodiments, the myostatin inhibitor is a polypeptide comprising a myostatin binding domain of a coco protein such as the human coco protein shown in FIG. 3 or GenBank Accession No. 2227329. Exemplary human coco proteins are:

It is.

The terms “antibody” and “antibody agent” are used interchangeably herein and refer to an immunoglobulin molecule obtained by in vitro or in vivo generation of a humoral response, including polyclonal and monoclonal antibodies. This term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized mouse antibodies), heteroconjugated antibodies (e.g., bispecific antibodies), and recombinant single chain Fv fragments (scFv). Also included. The term “antibody” also includes antigen-binding forms of antibodies (eg, Fab ′, F (ab ′) ₂ , Fab, Fv, rIgG, and inverted IgG).

  The term “antigen-binding fragment” includes any portion of an antibody that binds to a target epitope. An antigen-binding fragment can be, for example, a polypeptide comprising a CDR3 region, or other fragment of an immunoglobulin molecule that retains the affinity and specificity of a myostatin epitope.

“Specifically binds” refers to a preferential association in whole or in part with a ligand and a specific target molecule (ie, “binding partner” or “binding moiety”) for a composition lacking the target molecule. Contains a reference. Of course, it will be appreciated that some non-specific interaction may occur between the myostatin neutralizing antibody and other proteins. Nevertheless, specific binding can be distinguished as being mediated through specific recognition of the myostatin protein. Typically, specific binding results in a much stronger association between the antibody and myostatin protein than between the antibody and other proteins (eg, GDF11). Specific binding by an antibody to myostatin under such conditions requires an antibody that is selected for its specificity for a particular protein. (As opposed to K _d, K _a) affinity constant of the antibody binding site for monovalent antigens of the same type is at least 10 ⁷ M, usually at least 10 ⁸ M, preferably at least 10 ⁹ M, more preferably at least 10 ¹⁰ M , And most preferably at least 10 ¹¹ M. Various immunoassay formats are suitable for selecting antibodies that specifically react with myostatin. For example, solid phase ELISA immunoassays are routinely used to select monoclonal antibodies that specifically react with proteins. For a description of immunoassay formats and conditions that can be used to determine specific reactivity, see Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

  Using competitive binding format immunoassays, the cross-reactivity of the antibody with myostatin can be determined, for example, to identify whether the test antibody is a myostatin neutralizing antibody. For example, myostatin protein or a fragment thereof is immobilized on a solid support. A test antibody is added to the assay and competes for binding of TGF receptor (such as ActRII or ALK7) to the immobilized antigen. The ability of the test antibody to compete with the binding of TGF receptor to the immobilized myostatin antigen is compared.

  Similarly, cross-reactivity can be determined using competitive binding format immunoassays, eg, to determine the specificity of myostatin neutralizing antibodies. For example, the myostatin protein or its myostatin epitope is immobilized on a solid support. Epitopes of other proteins (such as GDF-11, Nodal, or BMP-4, or other proteins that have sequence homology with myostatin) are added to the assay to bind potential myostatin neutralizing antibodies to the immobilized antigen. Make it conflict. Compare the ability of the test peptide to compete with the binding of the potential myostatin neutralizing antibody to the immobilized myostatin antigen. Standard calculation calculates the percent cross-reactivity of potential myostatin neutralizing antibodies against other antigens. In certain preferred embodiments, the myostatin neutralizing antibody has less than 10% cross-reactivity with GDF-11. In other preferred embodiments, the myostatin neutralizing antibody has less than 1%, 5%, or 10% cross-reactivity with BMP-4.

III. Exemplary Cerberus and Coco Derivatives In certain embodiments, the myostatin inhibitor is at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% over the entire length of the human or mouse Kerberos protein. Or a Kerberos polypeptide that shares more sequence identity (see below).

In certain other embodiments, the myostatin inhibitor is a polypeptide comprising a Kerberos sequence obtained from human, mouse, or other species, or variants thereof, including the N-terminal truncated form of Kerberos. Full-length mouse and human Kerberos proteins are disclosed in US 2002/0164682 A1, SEQ ID NOs: 2 and 8, respectively, which are also disclosed below in NCBI reference sequence format:
Human Cerberus full-length protein:

  Residues 106-119 (any one of these residues can start the Kerberos derivative) and 241-267 residues (any one of these residues can terminate the Kerberos derivative) ) Is underlined.

Mouse Kerberos full-length protein:

  Residues 106-119 (any one of these residues can start the Kerberos derivative) and 241-272 residues (any one of these residues can terminate the Kerberos derivative) ) Is underlined. Note that the mouse protein is generally homologous to the human protein throughout the sequence, except for the additional 5 residues at the C-terminus. Thus, when a non-human Kerberos derivative is used, the residue number refers to the residue number corresponding to the human sequence.

  As noted above, in certain embodiments, preferred fragments of a human Kerberos derivative protein start at any of 106-119 residues (including residues at both ends) at the N-terminus and any after residue 241 Is a fragment that ends in

Also included are Kerberos-derived variant sequences, including mutants or variants of the wild-type myostatin binding domain that retain myostatin binding activity and optionally substantially lose BMP-4 binding. Variant sequences that do not have BMP binding affinity alter the selectivity of the inhibitor (eg, for GDF-11 or nodal binding, with preferential binding to one of the proteins. More selective for myostatin. Other binding properties for K-myostatin (K is also included), which is also a typical (higher affinity than wild-type) or more discriminatory (less affinity than wild-type truncated) binding to BMP-4. _d and / or K _on or K _off rate), or as a method to improve biodistribution or half-life in vivo or during storage.

  Although certain other Kerberos sequences based on homology searches in a database of identified proteins are described below, the variant Kerberos polypeptides may be derived from such proteins. Since these sequences are retrieved from public databases available on the Internet, as these databases are updated, additional homologues of proteins in other species may be obtained. In addition, other species of Kerberos proteins, particularly mammalian Kerberos proteins, are Ab-mediated in expression libraries that use PCR, low stringency hybridization, and antibodies that cross-react with Kerberos homologues identified in the target species. It can be easily obtained by standard molecular biological procedures such as screening.

  For example, sequence alignments using software such as DNAStar's MegaAlign (supra) can identify the most conserved regions within known members of a protein family. PCR can then be performed using degenerate oligos over such most conserved regions and template DNA from the target organism. In preferred embodiments, such conserved regions include a kinase domain and / or a ligand binding domain.

  These similarly conserved regions can be used to create probes for screening nucleic acid libraries under moderate to low stringency hybridization conditions (see definition section).

  Xenopus homologue: gi | 1513088.

  Trough homologue: FuguGenscan_32561 / SINFRUP00000076662.

  In certain embodiments, the myostatin inhibitor shares at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or more sequence identity over the entire length of the human or mouse Kerberos protein. A Kerberos polypeptide (described below).

  In certain other embodiments, the myostatin inhibitor is a polypeptide comprising a coco sequence obtained from human, mouse, or other species, or variants thereof, including the N-terminal truncated form of coco. Full length human coco protein is disclosed above.

  Various Kerberos and coco polypeptides can be prepared as fusion proteins. The fusion protein has one or more additional polypeptides that enhance one or more of in vivo stability, in vivo half-life, attachment / administration, tissue localization or distribution, protein complex formation, and / or purification. May include a portion. For example, the fusion protein can comprise an immunoglobulin Fc domain and / or a purified subsequence selected from an epitope tag, a FLAG tag, a polyhistidine sequence, and a GST fusion. The myostatin antagonist protein is one or more selected from glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids bound to lipid moieties, and amino acids bound to organic derivatizing agents. It may contain modified amino acid residues.

  A fusion protein or binding protein system (e.g., non-fusion covalent linkage by cross-linking) is also a second myostatin that is a polypeptide affinity reagent that selectively binds to myostatin and competes with ALK7 or ALK4 receptor binding. Inhibitor domains may be included. The affinity reagent may be an antibody agent. Antibody agents include, for example, recombinant antibodies; monoclonal antibodies; VH domains; VL domains; scFv; Fab fragments; Fab 'fragments; F (ab') 2; Fv; or disulfide-linked Fv, fully human antibodies or humanized It can be a chimeric antibody, or an antigen-binding fragment thereof. The affinity reagent is a peptide or scaffold peptide that selectively binds to myostatin and competes with ALK7 or ALK4 receptor binding. The affinity reagent may comprise a myostatin binding domain of ALK7 or ALK4. For example, the extracellular domain of ALK7 or ALK4 (preferably human ALK7 or ALK4) can be used. The affinity reagent may be a small organic molecule that selectively binds to myostatin and competes with ALK7 or ALK4 receptor binding.

Examples of human ALK7 myostatin binding domains are shown below:

Examples of human ALK4 myostatin binding domains are shown below:

  As shown herein, Caronte and thus human Cerberus and possibly Coco also do not substantially inhibit activin A signaling in the A204 reporter gene assay. As such, such myostatin antagonists are preferably considered to exhibit little interaction with activin A-mediated signaling.

IV. Exemplary therapeutic uses Full-length and N-terminally truncated Kerberos and coco derivatives, such as this variant coco and cerberus polypeptides, are useful for treating many diseases caused or exacerbated by the presence of myostatin. It can be used in the field of treatment.

  In certain embodiments, the polypeptides and derivatives thereof are used as part of a treatment for muscular dystrophy. The term “muscular dystrophy” refers to a group of degenerative muscular diseases characterized by gradual weakness and deterioration of skeletal muscle and possibly myocardium and respiratory muscle. Muscular dystrophy is an inherited disease characterized by progressive muscle atrophy and muscle weakness that begins with microscopic changes in muscle. As muscles degenerate over time, a person's muscle strength decreases. Exemplary muscular dystrophies that can be treated with therapies containing the present myostatin include the following: Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreis muscular dystrophy (EDMD), limb girdle Muscular dystrophy (LGMD), facial scapulohumeral muscular dystrophy (FSH or FSHD) (also known as Landsy Degerine), myotonic dystrophy (MMD) (also known as Steinert's disease), oropharyngeal muscular dystrophy (OPMD) ), Distal muscular dystrophy (DD), congenital muscular dystrophy (CMD).

  Duchenne muscular dystrophy (DMD) was first described by the French neurologist Guillaume Benjamin Amand Duchenne in the 1860s. Becker muscular dystrophy (BMD) is named after the German doctor Peter Emil Becker, who first described this variant of DMD in the 1950s. DMD is one of the most common genetic disorders in men, affecting 1 in 3,500 boys. DMD occurs when the dystrophin gene located on the short arm of the X chromosome is damaged. Since males have only one copy of the X chromosome, there is only one copy of the dystrophin gene. In the absence of dystrophin protein, muscles are easily damaged during the contraction and relaxation cycle. Muscles are supplemented by regeneration early in the disease, after which muscle progenitor cells are unable to respond to the progression of damage and healthy muscles are replaced with non-functional fibro-adipose tissue.

  In DMD, boys begin to show signs of muscle weakness at about 3 years of age. The disease gradually weakens the skeletal or voluntary muscles of the arms, legs, and trunk. By early teens or earlier, boys' myocardium and respiratory muscles can be affected as well. BMD is a much milder form of DMD. Its onset is usually in teens or early adults, and the course is slower than the course of DMD and much less predictable than the course of DMD. (DMD and BMD occur almost exclusively in boys, but rarely in girls. May develop).

  Until the 1980s, little was known about the cause of any type of muscular dystrophy. In 1986, a dystrophin gene deficiency was identified as the cause of DMD. BMD is caused by different mutations within this same gene. BMD patients have some dystrophin, but the amount is inadequate or of poor quality. Due to the presence of some dystrophin, the muscles of BMD patients are protected from severe or rapid degeneration like the muscles of DMD patients.

  Recent studies have demonstrated that blocking or eliminating myostatin function in vivo can effectively treat at least some symptoms of DMD and BMD patients (Bogdanovich et al., Supra; Wagner et al. ., Supra). Thus, the present Kerberos derivative, particularly its N-terminal truncated form, constitutes another means of blocking myostatin function in vivo in DMD and BMD patients.

  Similarly, the present coco or Kerberos derivative, particularly its N-terminal truncated form, provides an effective means of increasing muscle mass in other disease states that require muscle growth. For example, Gonzalez-Cadavid et al. (Supra) show that myostatin expression is inversely correlated with human lean mass index and that increased myostatin gene expression is associated with weight loss in patients with AIDS wasting syndrome. Reported. Inhibiting myostatin function in AIDS patients can alleviate at least some symptoms of AIDS, even if not completely eliminated, thus significantly improving the quality of life of AIDS patients.

  Since loss of myostatin function is also associated with fat loss without reduced nutrient intake (Zimmers et al., Supra; McPherron and Lee, supra), the coco or cerberus derivatives, especially its N-terminal truncated form, It can also be used as a therapeutic to delay or prevent the onset of obesity and type II diabetes.

  Cancerous anorexia-cachexia syndrome is one of the most debilitating and life-threatening aspects of cancer. Progressive weight loss in anorexia nervosa-cachexia syndrome is a common feature of many types of cancer and is associated with weight loss as well as poor quality of life and poor response to chemotherapy Causes a shorter survival time than seen in patients with no comparable tumor. Associated with anorexia, fat and muscle tissue atrophy, psychological distress, and poor quality of life, cachexia results from complex interactions between cancer and the host. Cachexia is one of the most common causes of death among cancer patients and is present at 80% at death. Cachexia is a complex example of metabolic disorders that affect protein, carbohydrate, and fat metabolism. Tumors produce direct and indirect abnormalities resulting in anorexia and weight loss. At present, there are no therapies that regulate or reverse this process.

  Cancerous anorexia-cachexia syndrome affects cytokine production, release of lipid mobilization factors and proteolytic inducers, and changes in intermediary metabolism. Anorexia is common, but a reduction in food intake alone cannot explain the changes in body composition seen in cancer patients, and an increase in nutrient intake cannot recover the wasting syndrome. Cachexia should be suspected in cancer patients if involuntary weight loss of more than 5 percent of pre-attack weight occurs within a 6-month period.

  Pharmaceutical composition since systemic overexpression of myostatin in adult mice was found to induce loss similar to the significant muscle and fat loss seen in human cachexia syndrome (Zimmers et al., Supra) The present coco and cerberus derivatives, particularly the N-terminal truncated form thereof, can be advantageously used as myostatin antagonists / blockers to prevent, treat, or alleviate symptoms of cachexia syndrome where muscle growth is desired.

  In certain embodiments, the variant coco or Kerberos polypeptide, particularly an N-terminal truncated Kerberos derivative, can be advantageously used to prevent, treat or alleviate a host condition of a disease involving neurodegeneration. Without wishing to be bound by any particular theory, the present Kerberos derivative can block the inhibitory feedback mechanism mediated through the wild-type ALK7 receptor, allowing the growth and differentiation of new neurons To do. This Kerberos derivative as a pharmaceutical composition prevents symptoms of diseases associated with neurodegeneration, including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease, etc. It can be advantageously used to treat or alleviate.

  Alzheimer's disease (AD) is a chronic, incurable, and uncontrollable central nervous system (CNS) disease that occurs gradually and results in memory loss, abnormal behavior, personality changes, and impaired thinking ability. These losses are associated with the death of certain types of brain cells and the disconnection between them.

  AD is described as a retrograde of early childhood development. Symptoms appear after 60 years in most AD patients. Initial symptoms include loss of recent memory, misjudgment, and personality changes. Later in the disease, AD patients can forget how to do simple tasks such as washing their hands. Eventually, AD patients lose all reasoning ability and become dependent on others for their daily care. Eventually, the disease becomes debilitating, patients become bedridden, and typically develop complications. AD patients most commonly die from pneumonia 8-20 years after the onset of the disease.

  Parkinson's disease (PD) is a chronic, incurable, and uncontrollable CNS disease that occurs gradually and results in uncontrollable physical movement, stiffness, tremor, and difficulty walking. These motor system problems are related to the death of brain cells in areas of the brain that produce dopamine, a chemical that helps regulate muscle activity.

  In most PD patients, symptoms appear after 50 years. The initial symptom of PD is a significant tremor that occurs in the limbs, especially the hands or lips. The subsequent characteristic symptoms of PD are stiffness or slowing of movement, drag walking, forward bending posture, and balance disorder. There are a wide range of secondary symptoms, including memory loss, dementia, depression, emotional changes, dysphagia, abnormal speech, sexual dysfunction, and bladder and bowel disorders. These symptoms begin to interfere with daily activities such as holding a fork or reading a newspaper. Eventually, PD patients become severely disabled and become bedridden. PD patients usually die of pneumonia.

  Amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease; motor neuron disease) is a chronic, incurable, and uncontrollable CNS disease that attacks motor neurons, a component of the CNS that connects the brain to skeletal muscle. . In ALS, because the motor neurons are altered and eventually die, the patient's brain usually continues to function and remain agile, but no motor commands reach the muscles.

  Most patients with ALS are between 40 and 70 years old. The first motor neurons that weaken are those that reach the arms or legs. ALS patients can have difficulty walking, dropping objects, falling, pronounced indefinitely, and laughing or crying out of control. Eventually, the limb muscles begin to atrophy because they are not used. This muscle weakness becomes debilitating and the patient needs a wheelchair or cannot function outside the bed. Most ALS patients die from ventilator-assisted complications resembling respiratory failure or pneumonia 3-5 years after the onset of the disease.

  The cause of these neurological diseases remains largely unknown. Although these have traditionally been defined as different diseases, they clearly show extraordinary similarity in the basic course and show far more overlapping symptoms than would be expected from chance alone. The current definition of disease does not adequately address the problem of duplication and a new classification of neurodegenerative diseases is required.

  Huntington's disease (HD) is another neurodegenerative disease caused by genetically programmed degeneration of neurons in specific areas of the brain. This degeneration results in uncontrolled movement, loss of intellectual ability, and emotional disturbance. HD is a familial disease transmitted from parents to children through dominant mutations in wild-type genes. Some early symptoms of HD are mood swings, depression, temper, or driving, learning new things, remembering facts, or difficulty in making decisions. As the disease progresses, it becomes increasingly difficult to concentrate on intellectual issues and patients can also have difficulty eating and swallowing on their own. The rate of disease progression and age of onset vary from person to person.

Tay-Sachs disease and Sandhoff disease are glycolipid storage diseases caused by the absence of lysosomal β-hexosaminidase (Gravel et al., In The Metabolic Basis of Inherited Disease, eds. Scriver et al., McGraw-Hill , New York, pp. 2839-2879, 1995). In any disease, G _M2 ganglioside and β- hexosaminidase associated glycolipid substrates accumulate in the nervous system and trigger acute neurodegeneration. In the most severe form, the onset of symptoms begins in early infancy. A rapid neurodegenerative course then follows, and affected infants exhibit motor dysfunction, seizures, vision loss, and hearing impairment. Death usually occurs by 2-5 years of age. Neuronal loss through the apoptotic mechanism has been demonstrated (Huang et al., Hum. Mol. Genet. 6: 1879-1885, 1997).

  It is well known that apoptosis contributes to the development of AIDS in the immune system. However, HIV-1 also induces neurological diseases. Shi et al. (J. Clin. Invest. 98: 1979-1990, 1996) examined apoptosis induced by HIV-1 infection of the central nervous system (CNS) in in vitro models and brain tissue of AIDS patients, We found that HIV-1 infection of primary brain cultures induced apoptosis in neurons and astrocytes in vitro. Neuronal and astrocyte apoptosis was also detected in brain tissues of 10/11 AIDS patients, including 5-5 HIV-1 dementia patients and 4/5 non-dementia patients.

  Neuronal loss is also a hallmark of prion diseases such as human Creutzfeldt-Jakob disease, bovine BSE (mad cow disease), sheep and goat scrapie disease, and cat feline spongiform encephalopathy (FSE) .

  The present Kerberos derivatives, including N-terminal truncated Kerberos derivatives, are also useful for preventing, treating, and alleviating symptoms of various PNS diseases as described below. The PNS is composed of nerves that lead to or branch off from the CNS. Peripheral nerves are responsible for various functions of the body, including sensory functions, motor functions, and autonomous functions. If an individual has peripheral neuropathy, the nerves of the PNS are damaged. Nerve damage can result from many causes, such as disease, physical injury, addiction, or malnutrition. These etiologies can affect afferent or efferent nerves. Depending on the cause of the injury, the nerve cell axon, its protective myelin sheath, or both can be damaged or destroyed.

  The term peripheral neuropathy encompasses a wide range of diseases in which nerves outside the brain and spinal cord—peripheral nerves—are damaged. Peripheral neuropathy can also be referred to as peripheral neuritis, or the term polyneuropathy or polyneuritis can be used when many nerves are involved.

  Peripheral neuropathy is a widespread disorder with many causes at the root. Some of these causes are common, such as diabetes, while others are extremely rare, such as acrylamide poisoning and certain genetic disorders. The most common worldwide cause of peripheral neuropathy is leprosy. Leprosy is caused by Mycobacterium leprae, a bacterium that attacks the peripheral nerves of affected patients. According to statistics collected by the World Health Organization, an estimated 1.15 million people worldwide have leprosy.

  In the United States, leprosy is extremely rare and diabetes is the most common known cause of peripheral neuropathy. In the United States and Europe, it is estimated that over 17 million people suffer from diabetes-related polyneuropathy. Many neurological disorders are idiopathic and no known cause can be found. The most common hereditary peripheral neuropathy in the United States is Charcot-Marie-Tooth disease, which affects approximately 125,000 people.

  Another well-known peripheral neuropathy is Guillain-Barre syndrome, which is a viral disease such as cytomegalovirus, Epstein-Barr virus, and human immunodeficiency virus (HIV), or Campylobacter jejuni And from complications associated with bacterial infections including Lyme disease. The global incidence is approximately 1.7 cases per 100,000 people annually. Other well-known causes of peripheral neuropathy include chronic alcoholism, varicella-zoster virus infection, botulism, and gray leukitis. Peripheral neuropathy may manifest as a primary symptom or may be due to another disease. For example, peripheral neuropathy is the only symptom of diseases such as amyloid neuropathy, certain cancers, or inherited neuropathies. Such diseases can affect the peripheral nervous system (PNS) and central nervous system (CMS), as well as other body tissues.

Other PNS diseases that can be treated with the present Kerberos derivative, particularly N-terminal truncated Kerberos, include the following: brachial plexus neuropathies (cervical and first thoracic nerve roots, nerve trunks, nerve bundles, and arms Disorders of peripheral nerve components of the plexus, clinical symptoms include upper limb pain, sensory abnormalities, muscle weakness, and sensory declines, including birth trauma and other trauma; organisms, neuritis, radiotherapy;. and other .Adams et al pathology might be related ^{to, Principles of Neurology, 6 th ed} , see Pp1351-2); diabetic neuropathy (diabetic Peripheral nerves, autonomic nerves, and cranial nerve disorders related to cerebral neuropathy, usually due to diabetic microvascular injury, including small blood vessels that fill the nerves (neurotrophic blood vessels). Considered relatively common The pathology includes third nerve palsy; mononeuropathy; polyneuropathy; diabetic muscular atrophy; painful polyneuropathy; autonomic neuropathy; and thoracoabdominal neuropathy. , Principles of Neurology, 6 ^th ed, pp1325); mononeuropathy (a disease or trauma involving a single peripheral nerve that is disproportionate to evidence of isolated or pervasive peripheral neuropathy. Multiple mononeuritis refers to a condition characterized by multiple isolated nerve injuries, including a variety of conditions including ischemia; trauma; compression; connective tissue disease; accumulated trauma disease; and other conditions. Neuralgia (severe or aching pain that occurs along the course or distribution of peripheral or cranial nerves); Peripheral nervous system neoplasms (neoplasms arising from peripheral nerve tissue, including neurofibromas; Schwann Granule cell types; and malignant peripheral gods Includes transsheath tumors, see DeVita Jr et al., Cancer: Principles and Practice of Oncology, 5 ^th ed, pp1750-1); nerve compression syndrome (internal or external, nerve or nerve root mechanical Compression, which can result in conduction block of nerve impulses, for example, due to myelin dysfunction or axonal loss Nerve and nerve sheath damage can occur due to ischemia; Neuritis (a general term for inflammation of peripheral or cranial nerves. Clinical manifestations include pain; sensory dysfunction; paresis; or sensory dullness); polyneuropathy (diseases of multiple peripheral nerves. Depending on the type of nerve affected (e.g. sensory, motor or autonomy), distribution of nerve damage (e.g. distal vs. proximal), mainly by affected nerve component (e.g. demyelinating versus axis) C), various forms are classified according to etiology or genetic patterns).

  In certain embodiments, the full-length coco or Kerberos polypeptide, or variant thereof, is used as part of a treatment for a disease or condition characterized by excessive or undesirable levels of BMP, as described below.

  Ectopic ossification of muscles, tendons and ligaments is a frequent problem faced by orthopedic surgeons. Hannallah et al. (J Bone Joint Surg Am. 2004 Jan; 86-A (1): 80-91) investigated the ability of noggin (a BMP [bone morphogenetic protein] antagonist) to inhibit ectopic ossification. did. Three different doses of noggin expressing muscle-derived stem cells inhibited ectopic ossification induced by BMP-4 expressing muscle-derived stem cells. Each of the three different doses of noggin-expressing muscle-derived stem cells also significantly inhibited ectopic ossification induced by demineralized bone matrix. All 11 animals with Achilles tendonectomy developed ectopic ossification at the site of injury in the control limb. In contrast, limbs treated with noggin-expressing muscle-derived stem cells had 83% reduction in ectopic ossification, and 8 out of 11 animals showed evidence of ectopic ossification by radiography. None (P <0.05). Thus, delivery of noggin mediated by muscle-derived stem cells can inhibit ectopic ossification caused by BMP-4, demineralized bone matrix, and trauma in animal models, and BMP inhibitors (noggin or It has been shown that gene therapy delivering (Cerberus) can be a powerful method of inhibiting ectopic ossification in target areas of the body. See also Glaser et al. (J Bone Joint Surg Am. 2003 Dec; 85-A (12): 2332-42).

  Osteoarthritis (OA) is a joint disease characterized by osteophyte development, fibrosis, and articular cartilage damage. The effects of exogenous transforming growth factor beta (TGFβ) isoforms and bone morphogenetic protein (BMP) suggest a role for these growth factors in the development of OA. Scharstuhl et al. (Arthritis Rheum. 2003 Dec; 48 (12): 3442-51) used adenovirus overexpression of TGFβ-antagonists and BMP antagonists to block TGF-β and BMP signaling. Inhibitors tested included secreted pan-specific TGF-β antagonists called mouse latency-related peptide 1 (mLAP-1), intracellular inhibitory Smad6 (BMP antagonist), and Smad7 (TGF-β / BMP inhibition) Factor). Intra-articular injection of papain resulted in increased protein expression of several TGF-β and BMP isoforms in the synovium and cartilage. When the adenovirus was transfected into the joint, the transgene was strongly expressed in the synovial coating. Overexpression of mLAP-1, Smad6, and Smad7 significantly reduced osteophyte formation compared to controls. Overexpression of Smad6 and Smad7 also significantly reduced synovial thickening. Furthermore, the secretory TGF-β inhibitor mLAP-1 increased PG reduction in articular cartilage. These results indicate a critical role of excess endogenous TGF-β and BMP in the development of osteophyte and synovial thickening, implying excess endogenous TGF-β and BMP in the development of OA. In contrast, blocking cartilage damage by endogenous TGF-β suggests a protective role for TGF-β in articular cartilage. Thus, the present coco or cerberus pharmaceutical composition can be used as a BMP antagonist to treat OA, including the occurrence of osteophytes and synovial thickening.

  In the analysis of normal ovarian surface epithelial (OSE) and ovarian cancer (OC) cells, Shepherd and Nachtigal (Endocrinology. 2003 Aug; 144 (8): 3306-14) observed BMP4 mRNA expression and primary OC cells were mature BMP4 Has been found to produce In addition, each member of the downstream signaling pathway was expressed in primary OSE and OC cells. Upon treatment with BMP4, Smad1 was phosphorylated and caused nuclear translocation in normal OSE and OC cells. Interestingly, the BMP target genes ID1 and ID3 were up-regulated 10-15 fold in primary OC cells compared to a 2-3 fold increase in normal OSE. The proliferation of some primary OC cells was not significantly altered by BMP4 treatment. However, long-term treatment of primary OC cells with BMP4 resulted in decreased cell density and increased cell spreading and adhesion. These data demonstrate the existence and putative function of BMP signaling in normal OSE and OC cells, and thus the present Kerberos pharmaceutical preparation can be used to control BMP4 signaling in the development of OC.

  Progressive ossification fibrodysplasia (FOP), a rare hereditary disability disorder characterized by ectopic bone formation, is associated with the development of bone morphogenetic proteins (particularly BMP-4) that are skeletal It is known to play a role in formation and is of particular interest for general drugs because gene antagonists to BMP-4 (such as Noggin) may be useful in preventing lamellar bone formation. See Blaszczyk et al. (Eur J Dermatol. 2003 May-Jun; 13 (3): 234-7). Thus, the present Cerberus therapeutics can also be used to treat FOP.

  Atherosclerosis is currently regarded as an inflammatory disease that preferentially occurs in arteries exposed to turbulent flow conditions, including oscillatory shear stress (OS), in bifurcated arteries. Sorescu et al. (J Biol Chem. 278 (33): 31128-35, 2003) is a mechanosensory inflammatory factor in which BMP4 plays an important role in the early stages of atherogenesis in an injury-prone area. It suggests that there is. Therefore, it is possible to modulate the BMP-4-induced inflammatory response at an early stage of atherogenesis in such a region using the present Kerberos therapeutic agent.

  In the cranial development process, the substructure of the cranial connective tissue causes intramembranous ossification to form the skull (cranium crown). As the calvaria develops over the brain, a fibrous suture is formed between the calvarial plates. Brain enlargement is coupled to calvarial growth through a series of tissue interactions within the cranial suture complex. Skull fusion or early cranial suture fusion results in abnormal head morphology, blindness, and mental retardation. Recent studies have demonstrated that a gain-of-function mutation in the fibroblast growth factor receptor (fgfr) is associated with a symptomatic form of skull fusion. Noggin, an antagonist of bone morphogenetic protein (BMP), is required for patterning of the neural trunk, somites, and skeleton of the embryo. Warren et al. (Nature. 2003 Apr 10; 422 (6932): 625-9) show that noggin is expressed postnatally in the patent mesenchymal suture mesenchyme, but the fused cranial suture suture It shows that it is not expressed in the mesenchyme and that noggin expression is suppressed by FGF2 and symptomatic fgfr signaling. Noggin misexpression prevents cranial suture fusion in vitro and in vivo, so symptomatic fgfr-mediated cranial fusion is the result of abnormally high BMP activity due to inappropriate down-regulation of noggin expression It is suggested. Thus, the present Kerberos therapeutics can be used to down-regulate BMP activity in order to prevent or treat such pathologies.

V. Exemplary Formulations The present compositions can be used alone or as part of a combination therapy with other compounds / pharmaceutical compositions.

  Soluble coco or Kerberos derivative therapeutics, including N-terminal truncated Kerberos derivative therapeutics for use in the present methods, include water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), Or it can be conveniently formulated for administration using a biologically acceptable medium such as a suitable mixture thereof. The optimum concentration of the active ingredient in the chosen medium can be determined experimentally according to procedures well known to medicinal chemists. As used herein, “biologically acceptable medium” includes any solvent, dispersion medium, etc. that are considered suitable for the desired route of administration of the pharmaceutical preparation. The use of such media for pharmaceutically active substances is well known in the art. Except insofar as conventional media or agents are incompatible with the activity of the therapeutic agent, its use in the pharmaceutical preparations of the present invention is contemplated. Their formulation, including suitable excipients and other proteins, is described, for example, in the book Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton, Pa., USA 1985). These excipients include “deposit formulations” for injection.

  The pharmaceutical formulations of the present invention treat animal compositions such as live stock (such as cattle, sheep, goats, pigs, and horses) or domestic animals (such as cats and dogs). Also included may be pharmaceutical preparations of coco or kerberus derivative therapeutics suitable for animal use.

  The method of the present invention can also be provided by a refillable or biodegradable device. In recent years, various sustained-release polymer devices have been developed and tested in vivo for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. Various biocompatible polymers, including biodegradable and non-degradable polymers (including hydrogels) can be used to form implants for sustained release of therapeutic agents at specific target sites.

  The pharmaceutical composition according to the invention may be administered as a single dose or as multiple doses. The pharmaceutical compositions of the invention can be administered as individual therapeutic agents or in combination with other therapeutic agents. The treatments of the invention can be combined with conventional therapies, which can be administered sequentially or simultaneously. The pharmaceutical compositions of the invention can be administered by any means that allows the coco or kerberus derivative to reach the target cell / tissue / organ. In some embodiments, the route of administration is from the group consisting of oral, intravesical, intravenous, intraarterial, intraperitoneal, local administration to the blood supply of the organ in which the target cells are present, or direct administration to the cells. The selected route of administration is included. Intravenous administration is the preferred method of administration. This can be achieved using an infusion pump.

  As used herein, the phrases “parenteral administration” and “parenteral administration” refer to methods of administration, generally by injection, other than enteral administration and topical administration, including but not limited to intravenous. Intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, intrathecal, intraspinal, and intrasternal injection And injection.

  As used herein, the phrases “systemic administration”, “systemic administration”, “peripheral administration”, and “peripheral administration” are used to refer to metabolism and other similar processes as they enter the patient's whole body. By administration, as provided, other than direct administration to the central nervous system, administration of a compound, drug, or other substance (eg, subcutaneous administration).

  These compounds are administered by powder, ointment, or drop, including oral, intravesical, such as nasal, rectal, vaginal, parenteral, intracisal, and buccal and sublingual, such as by spray. It can be administered to humans and other animals for therapeutic purposes by any suitable route of administration, including local administration.

  Regardless of the route of administration chosen, the compounds of the present invention and / or the pharmaceutical compositions of the present invention that can be used in an appropriate hydrated form are described below or other conventional methods well known to those skilled in the art. To a pharmaceutically acceptable dosage form as described by.

  The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention is effective to achieve the desired therapeutic response for the particular patient, composition, and method of administration without causing toxicity to the patient. The amount of active ingredient can be varied to obtain the correct amount.

  The dosage level selected will depend on the activity of the particular compound of the invention used, or its ester, salt, or amide, route of administration, administration time, excretion rate of the particular compound used, duration of treatment, the particular used Other drugs, compounds and / or substances used in combination with therapeutics, age, gender, weight, medical condition, overall health status and previous medical history of the patient being treated, and well-known in the medical arts Will depend on a variety of factors, including similar factors.

  A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start a dose of a compound of the invention used in a pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, The dosage can be gradually increased until it is achieved.

  In general, a suitable daily dose of a compound of the invention is the lowest dose of compound that is effective to obtain a therapeutic effect. Such an effective dose will generally depend on the factors described above. Generally, the intravenous, intraventricular, and subcutaneous dose of a compound of the present invention to a patient is about 0.0001 to about 100 mg / kg body weight / day.

  If necessary, the effective daily dose of the active compound is administered as a sub-dose of 2, 3, 4, 5, 6 or more, optionally in unit dosage form, administered separately at appropriate intervals throughout the day. May be.

  The term “treatment” is also intended to encompass prevention, treatment, and cure.

  Patients receiving this treatment are generally primates (especially humans) and other non-human mammals such as horses, cows, pigs, and sheep; and any in need thereof, including poultry and pets Is an animal.

  The compounds of the present invention can be administered by themselves or as a mixture with pharmaceutically acceptable carriers and also in combination with other antibacterial agents such as penicillins, cephalosporins, aminoglycosides, and glycopeptides Can be administered. Thus, combination therapy includes sequential, simultaneous and separate administration of the active compounds in a manner that upon subsequent administration does not completely eliminate the therapeutic effect of the initially administered active compound.

  When used in combination with certain formulations, the coco or kerberus derivatives can be effective soluble drugs. The therapeutic polypeptide can be provided as a fusion peptide with a second peptide that promotes solubility. For example, the Kerberos derivatives of the present invention can be provided as part of a fusion polypeptide with all or a fragment of an immunoglobulin hinge or Fc portion that can promote solubility and / or serum stability.

  The present invention also contemplates peptidomimetic sequences of the polypeptide derivatives described herein.

In general, the nomenclature used herein and the experimental procedures utilized in the present invention include molecular techniques, biochemical techniques, microbiological techniques, and recombinant DNA techniques. Such techniques are explained fully in the literature. For example, “Molecular Cloning: A Laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology ", John Wiley and Sons, Baltimore, Md. (1989); Perbal," A Practical Guide to Molecular Cloning ", John Wiley & Sons, New York (1988); Watson et al.," Recombinant DNA ", Scientific American Birren et al. (Eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); US Pat. Nos. 4,666,828; 4,683,202; 4,801,531; methods described in 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, JE, ed. (1994); “Current Protocols in Immunology” Volumes I-III Coligan JE, ed. (1994); Stites et al. (Eds), "Basic and Clinical Immunology" (8 ^th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (e ds), “Selected Methods in Cellular Immunology”, WH Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, eg, US patents 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; No. 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, MJ, ed. (1984); “Nucleic Acid Hybridization” Hames, BD, and Higgins SJ, eds. (1985); “Transcription Hames, BD, and Higgins SJ, eds. (1984); “Animal Cell Culture” Freshney, RI, ed. (1986); “Immobilized Cells and Enzymes” IRL Press (1986); “A Practical Guide to Molecular Cloning "Perbal, B., (1984), and" Methods in Enzymology "Vol. 1-317, Academic Press;" PCR Protocols: A See "Guide To Methods And Applications", Academic Press, San Diego, Calif. (1990); Marshak et al., "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996). All of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures in the references are considered well known in the art and are provided for the convenience of the reader. All the information contained in the references is incorporated herein by reference.

EXAMPLES Although the present invention has been described broadly herein, it is believed that the present invention will be more readily understood by reference to the following examples. The following examples are included solely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

Example 1. Sources of caronte and human cerberus proteins.
The human Kerberos sequence was cloned into a human CMV-derived expression vector. This construct was transiently transfected into HEK293 cells using polyethyleneimine (PEI). After culture, cells were harvested and conditioned medium was collected for purification.

  Caronte (from chicken) was obtained from R & D Systems (Minneapolis, Minnesota).

Example 2. Caronte binding to GDF-11.
GDF-11 is a closely related homolog of myostatin that controls neural processes. GDF-11 was immobilized on a BiaCore CM5 chip using standard amine coupling procedures. Follow-up: Caronte (200 μg / ml; R & D Systems) was injected onto a chip coupled with GDF-11. The trace in FIG. 4 shows the binding of Caronte to GDF-11.

Example 3. Caronte and human Kerberos that inhibit GDF-11 and myostatin mediated signaling.
An A-204 reporter gene assay was used to assess the effects of Caronte and Cerberus on signaling by GDF-11, myostatin, and activin A. Cell line: human rhabdomyosarcoma (derived from muscle). Reporter vector: pGL3 (CAGA) 12 (described in Dennler et al, 1998, EMBO 17: 3091-3100). See FIG. This vector is generally useful for factors that signal through Smad2 and Smad3 because the CAGA12 motif is present in the TGF-β responsive gene (PAI-1 gene).
Day 1: Divide A-204 cells into 48-well plates.
Day 2: A-204 cells are transfected with 10 ug pGL3 (CAGA) 12 or pGL3 (CAGA) 12 (10 ug) + pRLCMV (1 ug) and fugene.
Day 3: Add factors (diluted in medium + 0.1% BSA). Inhibitors should be preincubated with the factor one hour prior to addition to the cells. After 6 hours, the cells are rinsed with PBS to lyse the cells.

  This is followed by a luciferase assay. In the absence of inhibitor, activin A showed 10-fold enhancement of reporter gene expression and ED50-2 ng / ml. GDF-8: ED50: ~ 5 ng / ml, 15-fold promotion. GDF-11: 16-fold promotion, ED50: ~ 1.5 ng / ml.

  As shown in FIG. 6, in the A204 reporter gene assay, Caronte inhibits GDF-11 signaling. ActRIIA-Fc (“IIA muG2a”) fusions also inhibit GDF-11 signaling. As shown in FIG. 7, Caronte does not inhibit activin A in the A204 reporter gene assay. ActRIIA-Fc fusion (“IIA muG2a”), as expected, inhibits activin A signaling. Caronte is therefore a selective inhibitor of GDF-11 / myostatin and does not affect activin A signaling. This type of selectivity suggests that Caronte, Cerberus, and Coco have relatively few side effects when used as therapeutic agents. As expected, Cerberus behaved very much like Caronte and inhibited myostatin signaling. See FIG.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

FIG. 2 is a schematic diagram of a full-length and N-terminal truncated Kerberos showing a complete C-terminal Cys knot domain. The lower panel shows the predicted position of the Kerberos N-terminal deletion based on the crystal structure of Noggin bound to BMP-7. Schematic representation of the location of Kerberos to which Wnt, Nodal, and BMP bind. BMP-2 and highly related BMP-4 bind competitively with Kerberos, possibly in the same region. Other, more distantly related or unrelated proteins such as TGF-β1, EGF, and PDGF do not compete with BMP-4. N-terminal truncated Kerberos still binds to Xnr-1 (mouse nodal Xenopus homolog). Coco is a BMP inhibitor. (A) A nucleotide sequence of Xenopus laevis showing the nucleotide sequence including the coding region and the corresponding translated amino acid sequence. (B) Alignment at the amino acid level of Xenopus, puffer, human, and mouse Coco and other family members, Cerberus and Caronte. See Bell et al. (2003) Development 130 (7): 1381-1389, which is incorporated herein by reference. Shows caronte binding to GDF-11. Tracing shows that Caronte binds to GDF-11 on the BiaCore chip. GDF-11 was immobilized on a BiaCore CM5 chip using standard amine coupling procedures. Follow-up: Caronte (200 μg / ml; R & D Systems) was injected onto a chip coupled with GDF-11. A-204 reporter gene assay is shown. The figure shows the reporter vector: pGL3 (CAGA) 12 (described in Dennler et al, 1998, EMBO 17: 3091-3100). This vector is generally useful for factors that signal through Smad2 and Smad3 because the CAGA12 motif is present in the TGF-β responsive gene (PAI-1 gene). In the A-204 reporter gene assay, Caronte inhibits GDF-11 signaling. ActRIIA-Fc (“IIA muG2a”) fusions also inhibit GDF-11 signaling. Caronte does not inhibit activin A in the A-204 reporter gene assay. ActRIIA-Fc fusion (“IIA muG2a”), as expected, inhibits activin A signaling. In the A-204 reporter gene assay, both Kerberos and Caronte inhibit GDF-8 signaling.

Claims (53)

  A myostatin antagonist protein comprising a myostatin binding domain of a Cerberus / Dan / Gremlin polypeptide or variant thereof, wherein the myostatin antagonist protein binds to one or more of nodal and / or myostatin A pharmaceutical preparation that neutralizes it and is substantially free of pyrogens as is suitable for administration as a human or veterinary therapeutic.   2. The pharmaceutical preparation of claim 1, wherein the myostatin antagonist protein promotes muscle tissue growth.   2. The preparation of claim 1, wherein the myostatin binding domain is selected from the group consisting of a Kerberos sequence or variant sequence thereof, and a Coco sequence or variant sequence thereof.   2. The preparation of claim 1, wherein the myostatin antagonist protein has a reduced ability to neutralize BMP-4 as compared to the corresponding wild-type cerberus / dan / gremlin polypeptide. The myostatin binding domain is an N-terminal truncated derivative of a wild-type cerberus protein that includes: the derivative binds myostatin, GDF-11, and / or nodal, but does not substantially bind BMP-4 The preparation according to claim 4:
(a) a sequence starting from a position corresponding to any one of 106-119 residues of human cerberus; and
(b) A sequence ending at a position corresponding to an arbitrary residue after 240 residues of human Kerberos.   2. The preparation of claim 1, wherein the myostatin binding domain is encoded by a polynucleotide that hybridizes under high stringency conditions to a human Kerberos or human coco coding sequence. 2. The preparation of claim 1, wherein the myostatin antagonist protein binds myostatin with a _Kd of 1 _[ mu _] M or less. The myostatin antagonist protein has one additional polypeptide moiety that enhances one or more of in vivo stability, in vivo half-life, uptake / administration, tissue localization or distribution, protein complex formation, and / or purification. 4. The preparation of claim 3, which is a fusion protein comprising.   9. The preparation of claim 8, wherein the fusion protein comprises an immunoglobulin Fc domain.   9. The preparation of claim 8, wherein the fusion protein comprises a purified subsequence selected from an epitope tag, a FLAG tag, a polyhistidine sequence, and a GST fusion.   One or more of the myostatin antagonist proteins selected from glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids bound to lipid moieties, and amino acids bound to organic derivatizing agents 2. The preparation of claim 1 comprising modified amino acid residues.   2. The preparation of claim 1, wherein the myostatin antagonist protein does not substantially inhibit activin A signaling in the A204 reporter gene assay.   A polypeptide affinity wherein the myostatin antagonist protein is a fusion protein further comprising a second myostatin inhibitor domain, wherein the second myostatin inhibitor domain selectively binds to myostatin and competes with ALK7 or ALK4 receptor binding. 2. The preparation of claim 1 which is a reagent.   14. A preparation according to claim 13, wherein the affinity reagent is an antibody agent. 15. The antibody agent according to claim 14, wherein the antibody agent is a recombinant antibody; monoclonal antibody; _VH domain; _VL domain; scFv; Fab fragment; Fab 'fragment; F (ab') ₂ ; Fv; or disulfide-bonded Fv. Preparation.   16. The preparation of claim 15, wherein the antibody agent is a fully human antibody or a humanized chimeric antibody, or an antigen-binding fragment thereof.   14. The preparation of claim 13, wherein the affinity reagent is a peptide or scaffold peptide that selectively binds to myostatin and competes with the binding of the ALK7 or ALK4 receptor.   14. A preparation according to claim 13, wherein the affinity reagent is the myostatin binding domain of ALK7 or ALK4.   14. The preparation of claim 13, wherein the affinity reagent is a small organic molecule that selectively binds to myostatin and competes with the binding of the ALK7 or ALK4 receptor. Pharmaceutical preparations suitable for use in mammals, including:
(1) a vector comprising the coding sequence of the myostatin antagonist protein according to any one of claims 1 to 12;
(2) a transcriptional regulatory sequence that provides for expression of the myostatin antagonist protein in vivo in an amount effective to promote growth of the mammalian muscle tissue; and
(3) A pharmaceutically acceptable carrier.   Myostatin binding of a Kerberos / Dan / Gremlin polypeptide or variant thereof that binds to and neutralizes one or more of nodal and / or myostatin to prepare a drug that promotes muscle tissue growth in human patients Use of a myostatin antagonist protein comprising a domain.   Of a Kerberos / Dan / Gremlin polypeptide or variant thereof that binds to and neutralizes one or more of nodal and / or myostatin for the preparation of a medicament that promotes the growth of muscle tissue in non-human mammals Use of a myostatin antagonist protein comprising a myostatin binding domain.   A method for inhibiting myostatin signaling in muscle or adipose tissue cells of an animal comprising administering a pharmaceutical preparation according to claim 1.   The pharmaceutical preparation is administered, at least in part, in an amount effective to reduce the severity of the pathological condition characterized by an abnormal amount, development, or metabolic activity of muscle or adipose tissue in the subject. 23. The method of claim 22, wherein   24. The method of claim 22, wherein the pharmaceutical preparation is administered in an amount effective to prevent, ameliorate, or reduce the severity of debilitating disease.   25. The method of claim 24, wherein the wasting disease is selected from the group consisting of aging wasting, cachexia, anorexia, DMD syndrome, BMD syndrome, AIDS wasting syndrome, muscular dystrophy, and neuromuscular disease.   23. The method of claim 22, wherein the pharmaceutical preparation is administered in an amount effective to prevent, ameliorate, or reduce the severity of metabolic disease.   27. The method of claim 26, wherein the metabolic disease is selected from the group consisting of obesity or type II diabetes.   21. A method of inducing adipogenic differentiation in an animal comprising administering a pharmaceutical preparation according to any one of claims 1-20.   30. The method of claim 28, wherein the method is used to reduce a subject's body fat ratio.   21. A method for promoting the growth of muscle tissue in an animal comprising the step of administering a pharmaceutical preparation according to any one of claims 1-20.   21. A method of treating or preventing congestive heart failure comprising administering to a patient in need thereof an effective amount of the pharmaceutical preparation of any one of claims 1-20.   21. A method of reducing age related frailty comprising administering to a patient in need thereof an effective amount of the pharmaceutical preparation of any one of claims 1-20.   21. A method of increasing bone density or promoting fracture repair in a subject comprising administering an effective amount of the pharmaceutical preparation of any one of claims 1-20.   21. A method of reducing a protein catabolism in a subject comprising administering an effective amount of the pharmaceutical preparation of any one of claims 1-20.   21. A method of treating or reducing the severity of muscular dystrophy in a patient comprising administering an effective amount of the pharmaceutical preparation of any one of claims 1-20.   Muscular dystrophy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifis muscular dystrophy (EDMD), limb girdle muscular dystrophy (LGMD), facial scapulohumeral muscular dystrophy (FSH or FSHD) (Landy Degerine ), Myotonic dystrophy (MMD) (also known as Steinert's disease), oropharyngeal muscular dystrophy (OPMD), distal muscular dystrophy (DD), congenital muscular dystrophy (CMD), congenital myoto (MC) ), Congenital paramyotonia (PC), central core disease (CCD), nemaline myopathy (NM), tubule myopathy (MTM or MM), and periodic paralysis (PP). 35. The method according to 35.   21. A method of treating or reducing the severity of a motor neuron disease in a patient, comprising administering an effective amount of the pharmaceutical preparation of any one of claims 1-20.   Motor neuron diseases include amyotrophic lateral sclerosis (ALS) (also known as Lou Gehrig's disease), infant progressive spinal muscular atrophy (SMA, SMA1, or WH) (type 1 SMA, Weldnig Hoffmann Also known as intermittent spinal muscular atrophy (SMA or SMA2) (also known as type 2 SMA), juvenile spinal muscular atrophy (SMA, SMA3, or KW) (type 3 SMA, Kugelberg Selected from the group consisting of medullary muscular atrophy (SBMA) (also known as Kennedy disease and X-linked SBMA), and adult spinal muscular atrophy (SMA) 37. The method according to 37.   21. A method of treating or reducing the severity of inflammatory muscle disease in a patient comprising administering an effective amount of the pharmaceutical preparation of any one of claims 1-20.   40. The method of claim 39, wherein the inflammatory myopathy is selected from the group consisting of dermatomyositis (PM / DM), polymyositis (PM / DM), and inclusion body myositis (IBM).   21. A method of treating or reducing the severity of neuromuscular junction disease in a patient comprising administering an effective amount of the pharmaceutical preparation of any one of claims 1-20.   42. The method of claim 41, wherein the neuromuscular junction disease is selected from the group consisting of myasthenia gravis (MG), Lambert Eaton syndrome (LES), and congenital myasthenia syndrome (CMS).   21. A method of treating or reducing the severity of a muscular disease resulting from an endocrine abnormality in a patient, comprising administering an effective amount of the pharmaceutical preparation of any one of claims 1-20.   44. The method of claim 43, wherein the muscle disease resulting from an endocrine abnormality is selected from the group consisting of hyperthyroid muscle disease (HYPTM) and hypothyroid muscle disease (HYPOTM).   21. A method of treating or reducing the severity of peripheral neurological disease in a patient comprising administering an effective amount of the pharmaceutical preparation according to any one of claims 1, 2, 3, or 20.   46. The method of claim 45, wherein the peripheral neurological disease is selected from the group consisting of Charcot-Marie-Tooth disease (CMT), DeJerrine Sottas disease (DS), and Friedreich schizophrenia (FA).   21. A method of treating or reducing the severity of a metabolic disease in a patient comprising administering an effective amount of the pharmaceutical preparation of any one of claims 1-20.   Metabolic diseases include phosphorylase deficiency (MPD or PYGM), acid maltase deficiency (AMD), phosphofructokinase deficiency (PFKM), debranching enzyme deficiency (DBD), mitochondrial myopathy (MITO), carnitine deficiency (CD), carnitine palmitoyltransferase deficiency (CPT), phosphoglycerate kinase deficiency (PGK), phosphoglycerate mutase deficiency (PGAM or PGAMM), lactate dehydrogenase deficiency (LDHA), and myoadenylate deaminase 48. The method of claim 47, wherein the method is selected from the group consisting of deficiency (MAD).   A variant Kerberos polypeptide that differs in one or more amino acid residues from a wild-type cerberus protein, and binds to and neutralizes one or more of nodal and / or myostatin; and (ii) An abnormal amount of muscle tissue or adipose tissue in a subject, at least in part, including treating the subject with a variant Kerberos polypeptide that has a reduced ability to neutralize BMP-4 compared to a wild-type Kerberos protein A method of reducing the severity of a pathological condition characterized by development, or metabolic activity.   The object includes simultaneous administration of one or more other compounds selected from the group consisting of: inhibiting bone resorption, stimulating bone formation, increasing bone mineral density. 49. A method according to any one of -48.   49. The method of any one of claims 22-48, comprising co-administration of bisphosphonate.   1 selected from the group consisting of glutamate antagonists, polypeptide growth factors, drugs that increase production of neurotrophic factors, anti-inflammatory agents, caspase inhibitors, protein kinase C inhibitors, vitamin E, coenzyme Q10, and creatine 49. The method of any one of claims 22-48, comprising co-administration of one or more other compounds.

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